Protein-polymer conjugates are an important class of molecules with broad industrial applications and include nearly 30 FDA-approved pharmaceuticals. However, a lack of experimental structure-function data prevents the rational design of conjugates with desired properties. Motivated by this need, we investigated a conjugate of the human galectin-3 carbohydrate-recognition domain (Gal3C) with the pharmaceutically-relevant polymer polyethylene glycol (PEG). A Gal3C variant containing an extrinsic cysteine was conjugated with PEG (Gal3C[T243C]-PEG), resulting in a conjugate that could be produced at sufficient scale for structural and biophysical analyses and purified to homogeneity. Overall, Gal3C[T243C]-PEG featured enhanced binding affinity for the native Gal3C ligand. In thermal unfolding experiments monitored by circular dichroism, Gal3C showed cooperative unfolding behavior with a single transition typical for globular proteins. Surprisingly, Gal3C[T243C]-PEG undergoes two transitions during thermal unfolding, the first with a temperature near that observed for Gal3C, and then a second at a much higher temperature. Mutagenesis studies which sequentially removed surface lysines from Gal3C[T243C]-PEG were also conducted to test the effect of surface charge on PEG-dependent protein stability and dynamics. We investigated the structure of the Gal3C[T243C]-PEG constructs using nuclear magnetic resonance (NMR) spectroscopy in aqueous solutions. [15N,1H]-HSQC spectra of Gal3C[T243C]-PEG were similar to Gal3C, indicating PEGylation did not perturb the global protein fold. Site-specific chemical shifts and line broadening observed in the Gal3C[T243C]-PEG HSQC spectra pointed to specific residues affected by PEGylation. NMR measurements of the diffusion properties of Gal3C[T243C]-PEG demonstrated that the protein and polymer do not behave as two independent subunits; however, NMR hydrogen-to-deuterium exchange experiments showed that Gal3C interactions with PEG were not persistent over long timescales. Overall, these observations provide critically needed experimental data that probe the roles of both temperature and polymer conjugation on the structure, function, and dynamics of proteins.

Biophysical structure-function relationships of a site-specifically PEGylated protein with enhanced binding function and thermal stability

ACS Spring 2022

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